Members of cdc 2 family of protein kinases are known for their pivotal role in the regulation of the eukaryotic cell cycle. The potential roles of neuronal specific cdc2-like kinases in stabilizing the neurofilament skeleton and in axonal morphogenesis through phosphorylation of neurofilament and tau are not well delineated. The main objectives of this project are (1) to disrupt the genomic locus of neuronal cdc2-like kinase (cdk5) in embryonic stem cells and then use the targeted cells to generate mouse models to study in vivo function of these kinases; and (2) to overexpress cdk5 in neuronal cell lines and in vivo in mice. We have characterized cdk5 genomic clones isolated from 129/svj library. A gene targeting construct has been engineered by deletion of exons 3 through 5 and insertion of neomycin resistance gene at the site of deletion. At the 3' end of the construct, herpes virus thymidine kinase gene is included for positive/negative selection using G418 and gancyclovir. We have also isolated a murine cDNA clone using RT PCR technique and obtained complete nucleic acid sequence data. We have successfully disrupted the cdk5 locus in ES cells and generated F1 heterozygous mice. In addition, we have obtained cdk5 null ES cells.